In order to effectively colonize a host and cause disease, bacterial pathogens must have the means to sense and respond to rapidly changing environmental conditions. The adaptable nature of these microbes is largely attributed to the activation or inhibition of proteins that directly or indirectly modulate gene transcription. However, bacteria also encode many regulatory small RNA (sRNA) molecules that can profoundly affect bacterial responses and virulence potential. Most of these sRNAs act by base pairing with target mRNA transcripts, allowing for the post-transcriptional induction or repression of gene expression. In wild type E. coli strains, including E. coli pathogens that cause disease either within or outside of the gastrointestinal tract, sRNAs are likely key regulators of stress resistance, colonization and persistence, and virulence, but the functional roles of sRNAs within these medically and economically important microbes have received slight attention. Productive interactions between individual sRNAs and target transcripts often require input from the RNA chaperone Hfq. Recently, we found that Hfq is critical to the fitness and virulence potential of extraintestinal pathogenic Escherichia coli (ExPEC). These pathogens cause an array of diseases including, sepsis, neonatal meningitis, and urinary tract infections, the latter of which rank among the most common of infectious diseases. Our findings concerning Hfq implicate sRNAs in ExPEC pathogenesis and provide an impetus for better defining the functionality of these regulatory molecules during infection. The primary objectives of this R21 application are to identify the spectrum and functional attributes of sRNAs that are expressed by ExPEC in response to relevant environmental stresses both in vitro and in vivo during the course of an infection. Results obtained from these studies should enhance our understanding of the how ExPEC manages to colonize host tissues and cause disease in the face of numerous innate defenses, highlighting novel targets for the development of both preventive and therapeutic treatments of ExPEC-induced infections. PUBLIC HEALTH RELEVANCE: Strains of Extraintestinal pathogenic Escherichia coli (ExPEC) cause an array of serious illnesses that affect several million individuals each year, costing billions in health care and time loss at work. We propose that ExPEC utilize small regulatory RNA molecules to adapt to rapidly changing environmental conditions during the course of an infection, enabling these pathogens to better colonize and persist within the host.